Although many studies have dealt with the subject of calcium (Ca) and irreversible myocardial cell damage, it is unclear whether cytosolic free Ca does rise during cell injury. Furthermore, if a rise in cell Ca is responsible for cell injury it must precede any signs of irreversible myocardial cell injury. The mechanisms responsible for cell injury will be studied in a cultured heart cell model by: 1. Manipulating cell conditions to promote injury and determining if a rise in cytosolic free Ca precedes any non-specific membrane permeability changes. The following manipulations will be used to promote cell damage; a) 02 and substrate deprication (when need pH will be decreased) with and without re-oxygenation, and b) addition of excess catecholamines. The extent of injury will be assessed by measuring the sarco-lemmal permeability to radioactive tracers, leakage into the supernatant of intracellular enzymes, and measurement of cell high energy phosphate compounds. To establish a casual role for Ca in irreversible cell injure the level of cytosolic free Ca will be measured to determine if it increases prior to the loss of membrane integrity. 2. Ascertaining whether there are conditions in which a rise in cytosolic free Ca can be dissociated from loss of membrane integrity. Previous results suggest that inhibition of the Na-K ATPase results in a large increase in total cell Ca with no leakage of intracellular enzymes. Does cytosolic free Ca rise significantly during pump inhibition? Also what happens to ionized cell Ca and membrane integrity if the Na-K pump is inhibited by K-free incubation and mitochondrial respiration is simultaneously blocked to prevent mitochondrial Ca uptake? Experiments of this type will allow an assessment of whether a rise in ionized cell Ca is always associated with cell damage. 3. Determining the source for the elevated cytosolic free Ca: release of mitochondrial Ca,decreased efflux of cell Ca secondary to a dissipated Na gradient, and Ca entry via Ca channels. In summary, the overall goal of the study is to establish condition which result in myocardial cell damage and determine the extend to which a rise in cell Ca is responsible for irreversible myocardial cell injury.